The invention relates to materials useful for bone tissue repair.
There have been a number of materials studied to initiate bone repair and/or to restore or replace missing bone to address the problem of stimulating formation of bone at specific sites.
Among the approaches used to address this problem is a conformational method whereby an implant material, usually made of metal ceramic or other inorganic material in a form intended to mimic the form of the missing bone, is inserted into the site in which bone replacement is required. There is a risk that the host will reject the material or there will be a failure of integration of the implant with normal skeletal tissue. Some ceramic materials such as ceramic tricalcium phosphate, although acceptably biocompatible with the host and bone, when used as an implant, appear to lack sufficient mechanical properties of bone for general utility and the bone does not consistently grow into and become incorporated within the implant.
Another approach involves substituting the missing bone tissue with a matrix which functions as a support into which the new bone growth can occur. The theory is that the matrix attracts the cells committed to an osteogenic pathway and the new bone grows in and through the matrix by the process referred to as osteoconduction. Allogeneic bone (non-host bone) grafts are used for this method, however there is a substantially high failure rate. Even when the allogeneic bone grafts are accepted by the host, healing periods for consolidation and capacity for mechanical stress are of comparatively long duration compared to autogeneic bone (host-bone) grafting. The use of allogeneic bone also presents the issue of transmissible viral agents.
A third method involves the process known as osteoinduction, which occurs when a material induces the growth of new bone from the host""s undifferentiated cells or tissues, usually around a temporary matrix. A number of compounds are shown to have such a capacity. See for example, U.S. Pat. Nos. 4,440,750 to Glowacki, 4,294,753 and 4,455,256 to Urist and 4,434,094 and 4,627,982 to Seyedin et al. The most effective of these compounds appear to be proteins which stimulate osteogenesis. However, when synthesized from natural sources they are present in extremely low concentrations and require large amounts of starting material to obtain even a minute amount of material for experimentation. The availability of such proteins by recombinant methods may eventually make the use of such proteins per se of more practical value. However, such proteins will probably still need to be delivered to the desired site in an appropriate matrix.
There have been compositions disclosed containing collagen and various forms of calcium phosphate directed to healing and bone growth.
U.S. Pat. No. 5,338,772 to Bauer et al. discloses a composite material containing calcium phosphate ceramic particles and a bio-absorbable polymer where the calcium phosphate ceramic is at least 50% by weight and the particles are joined by polymer bridges. The calcium phosphate ceramic particles are disclosed as having a size of about 20 microns to about 5 mm.
U.S. Pat. No. 4,795,467 to Piez et al. discloses a composition comprising calcium phosphate mineral particles admixed with atelopeptide reconstituted fibrillar collagen. The calcium phosphate mineral particles are disclosed as having a size in the range of 100-2,000 microns.
U.S. Pat. No. 4,780,450 to Sauk et al. discloses a composition for bone repair comprising particulate polycrystalline calcium phosphate ceramic, a phosphophorin calcium salt and a type I collagen in a weight ratio of 775-15:3-0.1:1. The ceramic particles are disclosed as being dense hydroxyapatite about 1 to 10 microns in diameter or larger dense hydroxy apatite ceramic particles of greater than about 100 microns in diameter.
PCT Application WO 94/15653 to Ammann et al. discloses formulations comprising tricalcium phosphate (TCP), TGF-xcex2 and, optionally, collagen. The TCP is disclosed as being a delivery vehicle for the TGF-xcex2 such that the TCP is of the particle size greater than 5 microns and preferably greater than about 75 microns. The most preferred range for the size of the TCP granules is disclosed as being 125-250 microns.
PCT Application WO 95/08304 discloses polymineralic precursor particles of hydroxyapatite mixed with insoluble collagen. The particle size of the polymineralic precursor particles are in the range from 0.5 microns to 5 microns. The precursor minerals are converted to hydroxyapatite by hydrolysis, and this process, it is believed, fuses the mineral to form monolithic hydroxyapatite.
British Patent Specification 1,271,763 to FMC Corporation discloses complexes of calcium phosphate and collagen.
Methods are provided for preparing fibrous tissues or cartilage and for growing bone by introducing at a target site of repair a matrix which is porous, biodegradable, three-dimensionally fixed, has shape memory and maintains structural integrity and porosity after implant for a period sufficient to augment the tissue or bone replacement process. The matrix comprises insoluble mineralized biopolymer fibers and a water-soluble binder which is rendered insoluble by cross-linking. In one embodiment the matrix comprises mineralized fibrillar insoluble collagen, collagen derivative or modified gelatin, bound with a binder. In one embodiment, the minerals comprise calcium phosphate immobilized within the matrix. The resulting product is lyophilized, cross-linked, dried and sterilized to form a porous matrix. The matrix may be used as a tissue repair material and/or a delivery vehicle for biologically active factor. The matrix may be implanted for bone regeneration and will retain its porosity and physical integrity for a period of greater than fourteen days after implant.
The matrix is produced using a water-insoluble biodegradable biopolymer such as collagen, collagen derivative or modified gelatin. If gelatin is used, it will be modified to be insoluble in aqueous environments. The collagen may come from mineralized or unmineralized collagen sources, usually unmineralized collagen sources. Thus, the collagen may come from bone, tendons, skin, or the like, preferably Type I collagen which involves a combination of two strands of xcex12 and one xcex11 collagen chains. The collagen may be from a young source, e.g., calf, or a mature source, e.g., cow of two or more years. The source of the collagen may be any convenient animal source, mammalian or avian, and may include bovine, porcine, equine, chicken, turkey, or other domestic source of collagen. The insoluble collagenous tissue which is employed will normally be dispersed in a medium at an elevated pH, using at least about pH 8, more usually about pH 11-12. Commonly, sodium hydroxide is employed, although other hydroxides may be used, such as other alkali metal hydroxides or ammonium hydroxide.
Native collagen may be utilized in accordance with the present invention. Native collagen contains regions at each end which do not have the triplet glycine sequence. These regions (the telopeptides) are thought to be responsible for the immunogenicity associated with most collagen preparations. The immunogenicity can be mitigated by the removal of these regions to produce atelopeptide-collagen by digestion with proteolytic enzymes, such as trypsin and pepsin.
The concentration of collagen for mineralization will generally be in the range of about 0.1 to 10 weight percent, more usually from about 1 to 5 weight percent. The collagen medium will generally be at a concentrate of the base in the range of about 0.0001 to 0.1N. The pH is generally maintained during the course of the reaction in the range of about 10-13, preferably about 12.
Insoluble, fibrillar collagen is preferably used and can be prepared by routine methods. Typically, this can be accomplished with by first mixing with isopropanol (IPA), diethyl ether, hexane, ethyl acetate, or other suitable solvent, and separating the collagen. The pH is typically lowered to about 3, then cooled to about 4xc2x0 C., and allowed to swell. The resulting slurry may be homogenized until the desired viscosity is attained.
The homogenized slurry is mixed with solvent, agitated, and the pH is raised to about 7. The fibrillar collagen is separated, rinsed with deionized water, and lyophilized. To produce mineralized fibrillar collagen, the purified insoluble collagen fibrils may be homogenized, placed in a reactor where calcium chloride (typically, 0.05 m) and tribasic sodium phosphate (typically, 0.03 m) are introduced at a controlled rate with stirring. Sodium hydroxide is used to adjust pH at 11.0xc2x10.5 as needed during this process. After mineralization, the collagen is rinsed with deionized water or phosphate buffer, combined with the binder and the pH is adjusted within a range of 8.0xc2x12.0. A method of addition of phosphate and calcium ions is described in U.S. Pat. No. 5,231,169.
The calcium phosphate may contain other ions, such as carbonate, chloride, fluoride, sodium or ammonium. The presence of carbonate results in a product having the properties of dahllite (carbonated hydroxyapatite), while fluoride provides a product having the properties of fluoridated apatite. The weight % of carbonate will usually not exceed 10, while the weight of % of fluoride will usually not exceed 2. preferably in the range of 0 to 1. These ions may be present in conjunction with the calcium and/or phosphate source, so long as the ions are compatible and do not result in precipitation in the reagent solutions.
The rate of addition of the calcium and phosphate ions is generally about one hour and no more than about 72 hours in order to achieve the particle size of about 5 microns or less. Generally, the addition period is in the range of about 2 to 18 hours, more usually, in the range of about 4 to 16 hours. Mild temperatures are employed, usually not more than about 40xc2x0 C., preferably in the range of about 15xc2x0 to 30xc2x0 C. The weight ratio of the collagen to calcium phosphate mineral will generally be in the range of about 20:1 to 1:1, and typically will be about 7:3.
Other additives such as, non-collagenous proteins, factors or drugs, such as BMP""s, TGF-xcex2, calcitonin, antibiotics may be included in the matrix by adding to the collagen slurry, prior or subsequent to calcium and phosphate addition. The amounts of such additives will generally be in the range of about 0.0001 to 2 weight % based on the biopolymer used as the matrix, such as collagen.
The amount of collagen present in the mineralized product will generally be from about 95% to 30%, based on the weight of collagen fibers exclusive of the binder.
To form a porous, three-dimensionally fixed tissue repair matrix having shape memory, the mineralized biopolymer fibers are mixed with a binder.
Preferably, purified soluble collagen is used as the binder by first mixing soluble collagen with a solvent, such as isopropanol (IPA), and isolating the collagen. The pH is lowered to about 3.0, then, when the collagen is dissolved, the pH is raised to 5.0 washed twice with the solvent, rinsed with deionized water, sieved, and lyophilized.
Other binders which may be used include, but are not limited to, gelatin, polylactic acid, polyglycolic acid, copolymers of lactic and glycolic acid, polycaprolactone, carboxymethylcellulose, cellulose esters (such as the methyl and ethyl esters), cellulose acetate, dextrose, dextran, chitosan, hyaluronic acid, ficol, chondroitin sulfate, polyvinyl alcohol, polyacrylic acid, polypropylene glycol, polyethylene glycol, water soluble methacrylate or acrylate polymers.
To prepare the porous matrix, the preferred soluble collagen binder is added to a mineralized biopolymer slurry and blended. Preferably, insoluble collagen is the biopolymer and a proportion of about 10% (wt:wt) soluble to insoluble collagen is used. The pH is adjusted to 8.0xc2x12.0, as needed. When the desired level of blending is achieved, the dispersion is frozen at xe2x88x9220xc2x0 C. to xe2x88x9280xc2x0 C.
The frozen slurry is lyophilized. The porous matrix may be cross-linked to enhance physical stability, increase the resorption time of the matrix, ease the handling of the final product and render the water-soluble binder insoluble. The lyophilized matrix is preferably cross-linked using glutaraldehyde in solution (typically, 0.01%) or vapor. If a solution is used, after removal of excess reagent, the matrix is dehydrated by lyophilization.
The porous matrix may also be formed by filtering the slurry of mineralized collagen fibers and binder to form a web. The dried web may then be cross-linked.
The porous structure may also be achieved by mixing the biopolymer collagen fibers, binder and leachable particles (soluble salts, such as sodium chloride) and/or high vapor pressure solids which can be later removed by sublimation. The slurry can be dried, then the leachable or sublimable particles can be removed to form the porous structure. The porous matrix may be cross-linked.
Other benefits of a cross-linked matrix include greater implant residence time and shape retention (no fragmentation of the implant). The matrix has a shape memory, meaning that it is compressible from its initial size, shape and porosity and can return from a compressed state to its initial size, shape and porosity. Moreover, this can occur without substantial loss of its fibers or binder. The matrix will also maintain its porosity in physical integrity after implantation for greater than about fourteen days particularly when used for bone growth.
Other cross-linking methods and agents may be used, such as formaldehyde, chromium salts, di-isocyanates, carbodiimides, difunctional acid chlorides, difunctional anhydrides, difunctional succinimides, dibromoisopropanol, epichlorohydrin, diepoxides, dehydrothermal cross-linking, UV radiation when dry, or E-beam or gamma radiation in aqueous solution.
Final product sterilization may be accomplished using gamma radiation, E-beam radiation, dry heat or ethylene oxide.
An advantage of the present invention is that the collagen fibrils and the immobilized calcium phosphate mineral form a matrix particularly advantageous for the replacement or augmentation of bone. The matrix maintains its physical integrity and its porosity for a period of greater than about fourteen days after implant into a physiological environment in the case of bone replacement. By physical integrity it is meant that the matrix also maintains its porosity which is important to the tissue replacement or augmentation process. The matrix also has shape memory as described above. This is advantageous in that the matrix can be compressed into a delivery vehicle such as a cannula and the delivery vehicle can be introduced at the site of desired tissue growth. By releasing the matrix from the delivery vehicle at the site, the matrix returns to its initial size, shape and porosity without substantial loss of its fibers and binder. This is in contrast to compositions which, immediately or shortly after implant, collapse into an amorphous, non-porous mass and lose the mineral.
The matrix according to the present invention will eventually biodegrade or be resorbed, so the porosity and physical integrity cannot be maintained beyond that limiting period. This process normally takes on average, over about 2 to 12 weeks, and is of course dependent upon the size of the matrix that is implanted. However, as long as the period after which there has been complete absorption or biodegradation of the matrix has not occurred prior to the bone replacement or augmentation process, the rate of biodegradation will be sufficient.
It is an aspect of the present invention that the calcium phosphate minerals, typically present as hydroxyapatite, are immobilized on the matrix, as opposed to being freely mobile throughout the matrix. It has been found that the calcium phosphate mineral according to the present invention is immobilized within the matrix. The cellular response can be altered in that phagocytic cells such as giant cells and macrophages are more prominent around particulate materials, frequently forming granulomas. Particles small enough to be phagocytized, approximately 3 to 5 microns or less in size, are taken up by phagocytic cells which further stimulate a localized tissue reaction. For example, it is observed during bone healing that particulate wear debris associated with artificial joints are found in the macrophages of adjacent tissue and is associated with increased bone resorption in animal models in a dose dependent manner (xe2x80x9cMacrophage/particle interactions: effect of size, composition, and surface areaxe2x80x9d, Shanbhag A S et al., J. Biomed. Mater. Res. 28(1), 81-90 (1994)). It is thus an advantage of the invention that the immobilized calcium phosphate mineral is released over time as 5 micron or less particles, an ideal size to be taken up by phagocytic cells. It is a further advantage of the invention that any release of the calcium phosphate mineral particles is controlled, which is a result of mineral being immobilized within the matrix. The advantages of the particle size and immobilization are shown in Example III, below.
The matrix material has application as a tissue or cartilage repair, or osteoconductive bone grafting material for spinal fusion, filling bone defects, fracture repair and grafting periodontal defects. By combining the subject composition with an osteogenic material, such as autogenous bone or autologous aspirated bone marrow, or osteoinductive bone growth factors, BMP""s, calcitonin or other growth factors, bone induction and growth may be further augmented. The subject composition may also contain other additives such as drugs, and in particular antibiotics. The matrix may also provide a substrate to which growth factors may bind, so that factors produced by the host or externally introduced can concentrate at the matrix. The subject compositions find application in tissue or cartilage repair, fracture repair, maxifacial reconstruction, spinal fusion, joint reconstruction, and other orthopedic surgical uses.
The following examples are provided by way of illustration and are not intended to limit the invention in any way.